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Nitrogenase and Nitrous Oxide Reductase: Biomolecular Engineering of Complex Redox Enzymes (N-ABLE)
Date du début: 1 janv. 2013, Date de fin: 31 déc. 2017 PROJET  TERMINÉ 

"Nitrogenase and nitrous oxide reductase are complex metalloenzymes that are highly sensitive to dioxygen. They catalyze economically and ecologically important chemistry, but their function is not understood in full detail. We propose to analyze the mechanisms of these enzymes and elucidate the complex biogenesis of their intricate metal cofactors, the FeMo cofactor and the CuZ site, respectively. To this end we will combine protein biochemistry under anoxic conditions with the systematic, stepwise re-assembly of the structural genes and maturation factors of nitrogenase and nitrous oxide reductase into a heterologous expression system in Escherichia coli as a host system with a facultatively anaerobic lifestyle.This approach combines the principles of protein biochemistry and synthetic biology and aims at conveying the catalytic capacities of nitrogenase and nitrous oxide reductase to a new host. Once recombinant proteins can be produced we will use directed evolution under a selection conditions of dioxygen stress to increase the oxygen-tolerance of the enzymes and extend the usability of the expression plasmids. With nitrogenase, we will then aim at transferring the ability to fix dinitrogen to eukaryotic hosts and will start with the well-established Pichia pastoris. As a long-term goal, the expression of nitrogenase in plant cells will provide the basis for a new era of green biotechnology that does not rely on chemical nitrogen fertilization."